Evidence has accumulated that the voltage dependent anion channel (VDAC), located on the outer membrane of mitochondria, plays a central role in apoptosis. The involvement of VDAC oligomerization in apoptosis has been suggested in various studies. However, it still remains unknown how exactly VDAC supra-molecular assembly can be regulated in the membrane. Previous studies suggested the possible influence of various proteins on the formation of VDAC oligomers, but the important issue of the VDAC oligomeric state regulation by lipids has not been studied so far. Nevertheless, the effect of lipids on the oligomerization of several membrane proteins has been mentioned in the literature and in general, protein-lipid interactions are under extensive investigation. In the present work, I addressed the influence of lipids on VDAC oligomerization experimentally by reconstituting the fluorescently labelled VDAC in giant unilamellar vesicles (GUVs)—a chemically well defined, cell-free minimal model system. Fluorescence cross-correlation spectroscopy was performed to determine the oligomeric state of VDAC. I investigated the effect of important for apoptosis anionic lipids, phosphatidylglycerol and cardiolipin, on VDAC oligomerization. I demonstrated that phosphatidylglycerol significantly enhances VDAC oligomerization in the membrane, whereas cardiolipin disrupts VDAC oligomers. These results suggest that up- or down- regulation of these lipids in mitochondria during apoptosis can tune VDAC oligomerization in the membrane. Thus, this study sheds light on the role played by the above-mentioned lipids in the regulation of VDAC oligomerization during apoptosis and provides additional information on the molecular mechanisms of the programmed cell death. Another objective of this work was to investigate the partitioning of VDAC into liquid disordered or liquid ordered lipid phases. The existence of lipid domains or the lipid rafts in mitochondria and VDAC enrichment in these rafts is still under debate. Additionally, mitochondrial VDAC was recently found in the plasma membrane. The role of this VDAC is not known, however, it was shown to be located in caveolae (specialized lipid rafts) and play an important role in neuronal apotosis and Alzheimer’s disease. Therefore, VDAC partitioning to the lipid rafts is an interesting question for investigation. The possibility to reconstitute VDAC into minimal model systems–GUVs with phase separation, allowed to reveal the preferential partitioning of VDAC into liquid disordered lipid domain, which suggests either non-raft localization of VDAC or the requirement of the other factors for the recruitment of VDAC into lipid rafts.

In the recent years, a rapidly increasing amount of experimental data has been generated by high-throughput technologies. Despite of these large quantities of protein-related data and the development of computational prediction methods, the function of many proteins is still unknown. In the human proteome, at least 20% of the annotated proteins are not characterized. Thus, the question, how to predict protein function from its amino acid sequence, remains to be answered for many proteins. Classical bioinformatics approaches for function prediction are based on inferring function from well-characterized homologs, which are identified based on sequence similarity. However, these methods fail to identify distant homologs with low sequence similarity. As protein structure is more conserved than sequence in protein families, structure-based methods (e.g. fold recognition) may recognize possible structural similarities even at low sequence similarity and therefore provide information for function inference. These fold recognition methods have already been proven to be successful for individual proteins, but their automation for high-throughput application is difficult due to intrinsic challenges of these techniques, mainly caused by a high false positive rate. Automated identification of remote homologs based on fold recognition methods would allow a signi cant improvement in functional annotation of proteins. My approach was to combine structure-based computational prediction methods with experimental data from genome-wide RNAi screens to support the establishment of functional hypotheses by improving the analysis of protein structure prediction results. In the first part of my thesis, I characterized proteins from the Ska complex by computational methods. I showed the benefit of including experimental information to identify remote homologs: Integration of functional data helped to reduce the number of false positives in fold recognition results and made it possible to establish interesting functional hypotheses based on high con dence structural predictions. Based on the structural hypothesis of a GLEBS motif in c13orf3 (Ska3), I could derive a potential molecular mechanism that could explain the observed phenotype. In the second part of my thesis, my goal was to develop computational tools and automated analysis techniques to be able to perform structure-based functional annotation in a high-throughput way. I designed and implemented key tools that were successfully integrated into a computational platform, called StrAnno, which I set up together with my colleagues. These novel computational modules include a domain prediction algorithm and a graphical overview that facilitates and accelerates the analysis of results. StrAnno can be seen as a first step towards automatic functional annotation of proteins by structure-based methods. First, the analysis of long hit lists to identify promising candidates for further analysis is substantially facilitated by integration and combination of various sequence-based computational tools and data from functional databases. Second, the developed post-processing tools accelerate the evaluation of structural and functional hypotheses. False positives from the threading result lists are removed by various filters, and analysis of the possible true positives is greatly enhanced by the graphical overview. With these two essential benefits, fold recognition techniques are applicable to large-scale approaches. By applying this developed methodology to hits from a genome-wide cell cycle RNAi screen and evaluating structural hypotheses by molecular modeling techniques, I aimed to associate biological functions to human proteins and link the RNAi phenotype to a molecular function. For two selected human proteins, c20orf43 and HJURP, I could establish interesting structural and functional hypotheses. These predictions were based on templates with low sequence identity (10-20%). The uncharacterized human protein c20orf43 might be a E3 SUMO-ligase that could be involved either in DNA repair or rRNA regulatory processes. Based on the structural hypotheses of two domains of HJURP, I predicted a potential link to ubiquitylation processes and direct DNA binding. In addition, I substantiated the cell cycle arrest phenotype of these two genes upon RNAi knockdown. Fold recognition methods are a promising alternative for functional annotation of proteins that escape sequence-based annotation due to their low sequence identity to well-characterized protein families. The structural and functional hypotheses I established in my thesis open the door to investigate the molecular mechanisms of previously uncharacterized proteins, which may provide new insights into cellular mechanisms.

In Europa und den USA stellt das Mammakarzinom die häufigste Krebserkrankung der Frau dar. Zwei Signalwege scheinen bei der Persistenz des Brustkrebses von besonderer Bedeutung zu sein – der Wnt-Signalweg und der Syndecan-1-Signalweg. Der Wnt-Signalweg ist in über 50 % der Brustkrebsfälle aktiviert, wobei die Ursachen für diese Aktivierung noch nicht vollständig geklärt sind. Bisher konnten Studien zeigen, dass Progesteron sowohl in den kanonischen als auch nicht-kanonischen Wnt-Signalweg regulatorisch eingreift. Die Rolle der Gestagene wurde, im Gegensatz zu den Östrogenen, bei der Karzinogenese des Mammakarzinom längere Zeit kontrovers diskutiert. Neuere Studien belegen jedoch deren funktionelle Relevanz in frühen Stadien der Karzinogenese. Aufgrund dieser Erkenntnisse und ihrer mehrfach nachgewiesenen antitumoralen Wirkung rücken Antigestagen-wirkende Substanzen als Therapieansätze wieder in den Fokus der Forschung. Innerhalb dieses Teilprojekts sollte die Wirkung des Antigestagens RU-486 auf die Regulation der am Wnt-Signalweg beteiligten Proteine untersucht werden sowie dessen funktionelle Relevanz für Brustkrebszellen. Hierfür wurden drei verschiedene Brustkrebszelllinien, MCF-7, T-47D und MDA-MB-231, verwandt. Als Ergebnis dieser Untersuchungen konnte ein neuer Wirkmechanismus von RU-486 auf Brustkrebszellen belegt werden. Dabei hemmte RU-486 die Expression von WNT1 in MCF-7-Zellen. Für diese Zelllinie scheint WNT1 ein entscheidender Überlebensfaktor zu sein, da sie einerseits die höchste WNT1-Expression aufwies und andererseits mit einer gesteigerten Vitalität auf die WNT1-Behandlung reagierte. Weitere Versuche zeigten, dass RU-486 über die Hemmung der WNT1-Expression zu einer verminderten Vitalität der MCF-7-Zellen führte. An dieser antitumoralen Wirkung von RU-486 scheint sowohl der Progesteron- als auch der Glukokortikoidrezeptor beteiligt zu sein. Da der Wnt-Signalweg in einem Großteil der Brustkrebsfälle aktiviert ist, sind in diesem Zusammenhang weitere Untersuchungen zur Wirkung von Antigestagenen auf diesen Signalweg sinnvoll. Auch der Einsatz von HSP90-Inhibitoren, welche die Aktivierung der Steroidhormonrezeptoren hemmen, wäre in diesem Kontext ein möglicher Ansatz. Diese Substanzklasse wird derzeit präklinisch und klinisch untersucht. Das zweite Teilprojekt beschäftigte sich mit der Regulation von Syndecan-1 und dessen Auswirkung auf Brustkrebszellen und der Interaktion mit dem Osteoprotegerin. Die Rolle von Syndecan-1 beim Brustkrebs ist derzeit noch nicht vollständig geklärt. Jedoch konnten zahlreiche Studien den Einfluss dieses Proteoglykans auf physiologische und pathophysiologische Prozesse belegen. Aufgrund der vielfältigen Interaktionen von Syndecan-1 mit anderen Proteinen und dem Vorliegen zweier verschiedener Formen – membranständig und löslich, erlangt diese Thematik eine hohe Komplexität. Innerhalb dieser Arbeit sollte die Expression und Regulation von Syndecan-1 in den schon im ersten Teilprojekt verwandten Brustkrebszellen untersucht werden. Zur Behandlung wurden für die Therapie des Mammakarzinoms relevante Substanzen ausgewählt (Zoledronsäure, Dexamethason, Aromataseinhibitor). Nachfolgend sollte der Einfluss der Syndecan-1-Regulation auf das Zellschicksal sowie die Interaktion mit Osteoprotegerin, einem bedeutenden Regulator des Knochenstoffwechsels, untersucht werden. Die Analysen zeigten, dass alle drei Zelllinien Syndecan-1 exprimierten. Eine starke Induktion der Syndecan-1-Expression erfolgte bei MCF-7-Zellen nach Behandlung mit Dexamethason. Die Frage zur funktionellen Relevanz dieser Dexamethason-abhängigen Syndecan-1-Stimulation für das Zellschicksal dieser Brustkrebszelllinie blieb, trotz der umfangreichen Untersuchungen verschiedenster Funktionen (Vitalität, Apoptose, Migration und Invasion), unbeantwortet. Von besonderer funktioneller Bedeutung sind jedoch die Ergebnisse zur Syndecan-1/Osteoprotegerin-Interaktion. Aus der Literatur ist bereits bekannt, dass Osteoprotegerin über die Heparansulfatseitenketten von Syndecan-1 gebunden, internalisiert und abgebaut werden kann. Neu ist jedoch, dass auch auf transkriptioneller Ebene eine Regulation von Osteoprotegerin durch Syndecan-1 erfolgt. So konnte anhand von Untersuchungen innerhalb dieses Teilprojekts gezeigt werden, dass die Hemmung der Syndecan-1-Expression mittels siRNA mit einer gesteigerten Expression von Osteoprotegerin einhergeht. Aufgrund der Bedeutung von Osteoprotegerin für den Knochenstoffwechsel können weitere Untersuchungen zur Osteoklastogenese angeschlossen werden. Mittels Osteoklastenassays sollte untersucht werden, inwieweit die erhöhten Osteoprotegerinkonzentrationen im konditionierten Brustkrebszellmedium die Reifung der Osteoklasten hemmt. Ergebnisse dieser Untersuchungen könnten zum Verständnis beitragen, inwieweit die Syndecan-1-Osteoprotegerin-Wechselwirkungen zur Entstehung von Knochenmetastasen beitragen. Zusammenfassend lässt sich hervorheben, dass durch diese Arbeit neue Erkenntnisse über zwei für das Mammakarzinom bedeutende Signalwege, den Wnt- und Syndecan-1-Signalweg, erbracht wurden. Im ersten Teilprojekt konnte ein neuer Mechanismus der antitumoralen Wirkung von RU-486, welche über die Hemmung des Protoonkogens WNT1 erfolgt, auf Brustkrebszellen belegt werden. Im zweiten Teilprojekt wurde Dexamethason als ein Regulator von Syndecan-1 identifiziert. Des Weiteren konnte gezeigt werden, dass sich Syndecan-1 und Osteoprotegerin nicht nur auf Protein- sondern auch auf der transkriptionellen Ebene beeinflussen.

Aus E. coli bestehende probiotische Produkte wie Mutaflor (Ardeypharm, Herdecke) und Symbioflor 2 (SymbioPharm, Herborn) werden seit Jahrzehnten erfolgreich für die Behandlung gastroenterologischer Erkrankungen verwendet. Die Probiotika gelten aufgrund der langjährigen Erfahrung als sicher. Seit ca. 20 Jahren werden zunehmend Studien ins Leben gerufen, welche sowohl die Wirkung der Produkte klinisch bestätigen als auch die bisher unbekannten Wirkmechanismen aufklären sollen. Das in Mutaflor enthaltene Bakterium E. coli Nissle 1917 wurde bereits erfolgversprechend in klinischen Studien zur Remissionserhaltung bei Colitis ulcerosa getestet und wird seither als therapeutische Alternative zur Standardmedikation eingesetzt. Auch die Wirkung von Symbioflor 2 bei Erwachsenen und Kindern mit Reizdarmsyndrom konnte in ersten klinischen Studien belegt werden. Es gibt bereits zahlreiche Forschungsarbeiten mit E. coli Nissle 1917, die sich mit der molekularen Charakterisierung des Stamms befassen. Auch das Genom des Stamms wurde sequenziert. Dennoch fehlen schlüssige Argumente, welche Gene, Genprodukte und molekularen Mechanismen den probiotischen Effekt von EcN bewirken. Im Rahmen dieser Arbeit wurde nun das Produkt Symbioflor 2 näher untersucht. Es besteht aus den sechs E. coli-Genomotypen G1/2, G3/10, G4/9, G5, G6/7 und G8, die ursprünglich aus dem Habitat eines Spenders isoliert wurden. Alle sechs Genome inklusive der insgesamt zwölf natürlich enthaltenen Plasmide wurden sequenziert, annotiert und manuell nachbearbeitet. Die sechs E. coli-Genomotypen repräsentieren zusammen das im Produkt Symbioflor 2 enthaltene Pangenom. Somit konnten genomisch kodierte Virulenz- und Fitnessfaktoren analysiert werden. Ein Vergleich mit einer Vielzahl anderer bisher sequenzierter E. coli ermöglichte eine Einordnung der Symbioflor 2 E. coli in das Cluster der apathogenen E. coli. Unter Verwendung eines in vitro Testsystems mit humanen intestinalen Epithelzellen konnte gezeigt werden, dass die probiotischen Stämme E. coli Nissle 1917 und E. coli G3/10 im Gegensatz zu Kontrollstämmen die Adhärenz enteropathogener E. coli signifikant hemmen. In weiteren Versuchen konnten dann kleine ribosomal synthetisierte und antibakteriell wirksame Moleküle, in EcN die Mikrozine M und H47, für diesen Effekt verantwortlich gemacht werden. In der Folge wurde auch in E. coli G3/10 ein neues, bisher unbeschriebenes Mikrozin detektiert, welches Mikrozin S genannt wird. Zudem konnten vier Gene auf dem Plasmid pSYM1 lokalisiert werden, die unterschiedliche Funktionen bei der Produktion von Mikrozin S haben. Zwei der Gene kodieren am Transport beteiligte Proteine. Ein kleiner Leserahmen konnte als das Mikrozin S-kodierende Gen mcsS identifiziert werden. Ein weiteres Gen vermittelt eine Immunität gegenüber Mikrozin S. Seine Expression in einem zuvor sensitiven Stamm macht diesen resistent gegenüber der Wirkung von Mikrozin S. Erst im September 2011 erfolgte ein erster Eintrag in die NCBI-Datenbank, in dem die Gensequenz von mcsS plasmidkodiert in einer Shigelle annotiert als hypothetisches Protein aufgeführt ist. Dem Gen wurde keine Funktion zugewiesen. Wird die Expression von mcsS in dem E. coli-Laborstamm MDS42 in Abwesenheit eines Immunitätsproteins induziert, wirkt das Peptid toxisch auf die bakteriellen Zellen. Mikrozin S kann zudem anhand seiner Aminosäuresequenz und der genetischen Organisation in die Mikrozine-Klasse IIa eingeordnet werden. Mikrozine können vielfältig verwendet werden, haben jedoch im Vergleich zu Bakteriozinen Gram-positiver Bakterien bisher zu wenig Beachtung gefunden. Anwendungsmöglichkeiten liegen in der Lebensmittelindustrie, der Human- und Veterinärmedizin, wo Mikrozin S nah verwandte Gram-negative Bakterien im Wachstum hemmen bzw. abtöten könnte. Zum Beispiel wird im Rahmen dieser Arbeit die Wirkung von E. coli Nissle 1917 und E. coli G3/10 auf enterohämorrhagische E. coli in vitro getestet. Es wird gezeigt, dass das von E. coli G3/10 gebildete Mikrozin S eine Adhärenzminderung aller verwendeten EHEC-Stämme an humane intestinale Epithelzellen vermittelt. Da EcN nur einen der vier getesteten EHEC-Stämme inhibiert, wurden Untersuchungen begonnen, die die Ursache dafür thematisieren. Die in dieser Arbeit generierten Ergebnisse bilden die Grundlage für weitere Studien zu den in dem Produkt Symbioflor 2 enthaltenen E. coli. Zudem können umfangreiche Analysen von Mikrozin S, wie die Reinigung des Proteins, seine Produktion in großem Maßstab und die Testung von Anwendungsmöglichkeiten fortgeführt werden.

Apicobasal polarity is essential for epithelia formation and maintenance. Cell junctions, namely the zonula adherens in Drosophila melanogaster, are the morphological landmarks that define and distinguish the apical from the basal surface. This resulting compartmentalisation is key for the cell and consequently the epithelia. To maintain proper junctions, cells make use of several protein complexes and their interactions. Among these complexes, the Crumbs (Crb) network stands out. Mutations in Crumbs (crb11A22) lead to zonula adherens collapse, consequent loss of apical surface and disaggregation of the epithelia. However, the mechanisms behind this are not known and havenʼt been addressed using modern techniques such as live imaging. Several things came out of the dataset obtained from the FRAP experiments. Firstly, protein kinetics are better described when a double exponential fit curve is used, which raises the possibility that two cell processes might be involved in the recovery observed for the different markers. Another finding was the fact that the kinetics of some polarised protein markers is not the same in every region of the embryo. Distinct areas of the embryo with different morphogenetic activity levels show different kinetics for the same compartment marker. That was the case with SpiderGFP (whole plasma membrane marker) and SASVenus (apical plasma membrane marker) where τ2 was lower in the posterior region of the embryo which is characterised by intense cell movements resulting from convergence extension. DE-CadGFP (zonula adherens marker) and lacGFP (basolateral marker) behaved similarly in the whole embryo. This indicates that convergence extension shows different trafficking needs for the apical surface. In crb11A22, SpiderGFP kinetic spatial differences were not observed. τ2 in the anterior (low level of morphogenesis) is affected and similar to wild type τ2 levels in the posterior. This could pinpoint the fact that the epithelia disaggregation is a result of trafficking failure of apical components. Live imaging of DE-CadGFP in crb11A22 background revealed initial disaggregation in the anterior part of the embryo, which strengthens the idea that Crb is required for adherens junction stabilisation and maintenance.

Prokaryotic cell division is one of the most fundamental processes in biology, but the dynamics and mechanics are far from being understood. In many bacteria, FtsZ, a tubulin homologue assembles into a ring-like structure – Z-ring at precisely the middle of the cell. This accurate site selection is dependent on the Min proteins. Min D and MinE self-organise into waves in vitro, and oscillate pole to pole in vivo. MinC is thought to couple the Min oscillations to FtsZ by direct interaction. The mechanism of inhibitory action of MinC on FtsZ assembly is not known. Critical to the understanding of regulation of FtsZ by MinC and other proteins and its probable role in force generation is the organisation, structure and the dynamics of the Z-ring. Current models of the FtsZ filament organization in the Z-ring argue between two different structures – (i) short overlapping protofilaments with lateral interactions and (ii) few long annealed protofilaments with or without lateral contacts. Our observations of the characteristics of polymerization and turnover studies using fluorescence microscopy suggest that the FtsZ filament is a continuous and irresolute bundle. The results are consistent with a structure where the turnover happens throughout, and any specialised structure resulting in a GTP cap like structure can be ruled out. We show that the turnover rates and hydrolysis rates are similar arguing for a model in which subunit leaves as soon as it hydrolyses GTP. On the basis of crystal structures, we cloned the N-terminal of FtsZ, which acts as a C-terminal end capping fragment and is able to interact with monomers. The end-capping fragment, NZ can disassemble the FtsZ polymers, without influencing the GTPase activity, offering a comparable standard for the activity of MinC. On the basis of our observations, we propose a model on how MinC can disassemble FtsZ polymers. Furthermore, our data shows that the Min CDE system is sufficient to cause spatial regulation of FtsZ provided FtsZ is dynamic. How the Z-ring takes the form of a functional helical or ring-like structure remains unclear. Extensive modelling approaches have tried to explain the ring formation and force generation. Previous studies have qualitatively shown bending of liposome membranes by FtsZ filaments. We hypothesised that the presumably intrinsically curved filaments should respond to pre-curved substrates, and the alignment should be quantifiable. This should ascertain whether or not FtsZ has intrinsic curvature and/or actively induces any force. Thus, we investigated how FtsZ filaments respond to a range of curvatures, which mimic different stages of the division process. Our results show that the FtsZ filaments possess intrinsic curvatures as well as spontaneous twist. This facilitates the formation of Z-ring by utilizing geometrical cues. Our results are in agreement with consistent helical FtsZ polymers observed in vivo by Cryo-EM or super resolution microscopy. The alignment of filaments over a range of curvature suggests that the filaments have considerable flexibility, which strongly suggests reconsidering possible mechanisms of force generation. Moreover, the developed assay constitutes a valuable platform to further study proteins involved in modifying curvature of FtsZ filaments. In summary, by reconstituting the FtsZ filament in vitro, we have elucidated the nature of FtsZ filaments. The dynamics of FtsZ filaments allows them to be inhibited by MinC, thus cooperating with the Min waves. The presence of intrinsic curvature and twist facilitates their formation into a ring necessary for the cell to carry out cytokinesis.

Understanding the transcriptional mechanisms that control hematopoiesis and the interaction between hematopoietic stem cells and the bone marrow (BM) microenvironment in vivo is of considerable interest. The calcineurin-dependent transcription factor NFAT (Nuclear Factor of Activated T cells) is known as master regulator of cytokine production in T lymphocytes and therefore central for T cell-dependent immune reactions, but has also been shown to regulate a process of differentiation and tissue adaptation in various cell types. The activation of NFAT is dependent on the calcium level within the cell. In resting cells, calcium levels are low and NFAT is cytoplasmic and inactive. A sustained increase in the internal calcium concentration within an external stimuli leads to activation of the calcium-dependent calcineurin, followed by dephosphorylation and nuclear translocation of NFAT. We have previously shown that NFATc2, a member of the NFAT family, is expressed in CD34+ hematopoietic stem cells (HSC). A mouse model harboring NFATc2 deficiency provides the opportunity for in vivo investigation of the role of NFATc2 in hematopoiesis. Our recent observations showed that aged mice lacking the transcription factor NFATc2 develop peripheral blood anemia and thrombocytopenia, BM hypoplasia and extramedullary hematopoiesis in spleen and liver. The proliferation and differentiation of NFATc2-deficient hematopoietic stem cells ex vivo, however, was found to be intact. It remained therefore unclear whether the disturbed hematopoiesis in NFATc2-deficient mice was caused by the hematopoietic or the stroma component of the BM hematopoietic niche. In the current study we dissected the relative contribution of hematopoietic and stroma cells to the phenotype of the NFATc2-deficent mice by transplanting immuno-magnetically purified NFATc2-deficient (KO) HSCs to lethally irradiated wild type (WT) mice, and vice versa. After a post-transplantation period of 6-8 months, peripheral blood, BM as well as spleen and liver of the transplanted animals were analyzed and compared to WT and KO mice transplanted with control cells. Transplantation of NFATc2-deficient HSCs into WT recipients (KO WT) induced similar hematological abnormalities as those occurring in non-transplanted KO mice or in KO mice transplanted with KO cells (KO KO). Compared to WT mice transplanted with WT cells (WT WT), KO WT mice showed evidence of anemia, thrombocytopenia and a significantly reduced number of hematopoietic cells in their BM. Likewise, KO WT mice developed clear signs of extramedullary hematopoiesis in spleen and liver, which was not the case in WT WT control animals. In addition to the hematopoietic abnormalities, transplantation of NFATc2-deficient HSC also induced osteogenic abnormalities such as BM sclerosis and fibrosis in WT mice. This phenomenon was rather subtle and of incomplete penetrance, but never seen in mice transplanted with WT cells. These data demonstrate for the first time, that the NFATc2 transcription factor directly regulates the intrinsic function of hematopoietic stem cells in vivo. However, the transcriptional targets for NFAT in these cells are yet unknown. In addition to hematopoietic stem cells, NFATc2 has been shown to be expressed in a lineage-specific manner during myeloid differentiation and, notably, is maintained during megakaryopoiesis while it is suppressed during the differentiation of neutrophils. Bone marrow megakaryocytes are the precursors of peripheral blood platelets and therefore constitute an integral part of primary hemostasis, thrombosis and wound healing. The biological role of NFAT in megakaryocytes is unknown. We have recently shown that NFATc2 is not necessary for megakaryocytic differentiation. On the other hand, recent evidence suggests that NFATc2 is required for the transcription of specific megakaryocytic genes. In this study, we showed that activation of the calcineurin/NFAT pathway in either primary megakaryocytes or CMK megakaryocytic cells forces the cells to go into apoptosis. Cell death in megakaryocytes is induced by treating the cells with the calcium ionophore ionomycin and suppressed by either the pan-caspase inhibitor zVAD or the calcineurin inhibitor cyclosporin A (CsA). Ionomycin stimulation of megakaryocytes leads to the expression of Fas Ligand (FASLG), a pro-apoptotic member of the tumor necrosis factor superfamily. Expression of FASLG was detectable as early as four hours after stimulation on the membrane of ionomycin-treated megakaryocytes, was augmented in cells stably overexpressing NFATc2, and was suppressed in cells either pretreated with CsA or expressing the specific peptide inhibitor of NFAT, VIVIT. To investigate the physiological relevance of FASLG expression on megakaryocytes, we performed co-cultures of megakaryocytes with Fas-expressing T-lymphocytes, in which CMK cells were left either unstimulated or pre-stimulated with ionomycin and then added to Jurkat cells. The presence of ionomycin-stimulated CMK cells, but not of unstimulated cells or cells stimulated in the presence of CsA, significantly induced apoptosis in Jurkat cells. Overexpression of NFATc2 in CMK cells enhanced their potency to induce apoptosis in Jurkat cells, while cells expressing VIVIT were less effective. Apoptosis induction of Jurkat cells by stimulated CMK cells was partially blocked by the presence of either a neutralizing antibody against FASLG or an antagonistic antibody to Fas during the co-culture period, indicating involvement of the FASLG/Fas apoptosis pathway. These results represent the first clear evidence for a biological function of the calcineurin/NFAT pathway in megakaryocytes, namely the regulation of Fas/FASLG-dependent apoptosis. Second, they underline that the biological role of megakaryocytes is not restricted to the production of proteins and other cellular structures for platelet assembly, but that this population of cells fulfills an independent regulatory function in the context of the surrounding tissue. Finally, we have identified by RNA sequencing analysis of NFATc2-expressing and -deficient cells, the entire set of genes which is induced by NFATc2 in stimulated megakaryocytes. Functional pathway analysis suggests an involvement of NFATc2 in pro-inflammatory pathways in these cells. The significance of these findings has to be addressed in further studies.

This work was performed in the framework of an interdisciplinary graduate program that focuses on the establishment and extension of innovative compounds for the packaging of electronic systems. Such chemically or biotechnologically tailored compounds can be used for the direct patterning of optically, magnetically or biologically functional structures in nano- and biotechnical products. In order to organize matter at the nanometer scale, imprinting litho-graphy techniques or self-organization processes are appropriate. Fine-tuning of numerous engineering processes requires continuous and high precision monitoring as well as control of diverse parameters. These demands are only partially met by physical or chemical components since they use surrogate parameters, measure off-line, or provide insufficient performances. Biological compounds, in particular protein-based feedback systems, fulfill certain system requirements to a considerable degree. Hydrophobins and S-layer proteins are surface active proteins, produced by filamentous fungi or bacteria. In nature, these (self )assembly proteins form highly ordered and robust structures. In addition, their tolerance for different sequence manipulations and chemical modifications allows extensive functionalization of these nanometer-sized proteins. Hence, these surface active proteins can also be fused with other protein domains to create chimera, which retain function of both original proteins. In conclusion, both hydrophobins and S-layer proteins represent a versatile tool in numerous fields of applied biotechnology, medicine or diagnostics. But until now, efficient in vitro operation in molecular designed protein coatings is strongly restricted due to their complex assembly mechanism. In the first phase of this work, it was demonstrated, that representatives of class I and class II hydrophobins tend to form multilayered structures on solid surfaces. It was found that only two protein orientations seems to be preferentially formed. In the process of assembly, the orientation of the first hydrophobin layer strictly depends on the substrate wettability. Consequently, each of the following hydrophobin layers is inverse oriented to the layer before. This alternating assembly mechanism has to be taken into account, when working with functionalized hydrophobins, because a hydrophobin-fused functional protein domain is exclusively located on one side of the protein. Due to the densely packed structure of surface active proteins, a fused functional domain, embedded between two hydrophobins is barely available for external reagents. Basically, the simultaneous existence of a broad spectrum of ordered and disordered assembly structures, demonstrated the need of an uniform protein film assembly for applications in fine-diagnostics or biomedicine. With regard to molecular designed protein coatings, this work further aimed at establishing conditions to develop a method for a ‘layer-by-layer’ assembly of protein chimeras. Based on their amphiphilic character, self-assembly behavior of surface active proteins can be influenced by conventional ionic surfactants. In order to study the effect of surfactants on the composition and morphology of adsorbed protein films, contact angle measurements, nulling ellipsometry, SEM, AFM and AFAM were performed. It was found that the layer thickness of assembled protein films is strictly dependent on the amount of added surfactant. At certain threshold surfactant concentrations, hydrophobins and S-layer proteins assemble in uniform layers, which are as thick as expected for a protein monolayer or a bilayer. Assembled protein films are covered by a smooth surfactant layer, which prevents further protein assembly. AFAM measurements reveal the formation of well defined lattice structures under the coverage of surfactants. Even the removal of the surfactant layer is possible without inter-fering with protein specific secondary structures. Solvent accessibility and functionality of protein-fused domains was successfully demonstrated. As compared to conventional assembly techniques, this novel protein deposition method offers a possibility for a ‘directed’ protein coating on solid surfaces. In addition, it guarantees broadly ranged homogeneous assembly of protein chimeras on non-planar or even porous surfaces independent of their position. Finally, a prototype for an interfacial FRET was developed in a close collaboration with the Institute of Physical Chemistry (TUD). This innovative FRET between semiconducting nano-particles and illuminating protein chimeras takes place across an oil/water interface. Hydro-phobins were used to stabilize artificial oil droplets in aqueous solution. These small proteins possess the ability to attach fused functional domains very close to an oil/water interface. When, in addition to this, an optically active nanostructure directly docks to the hydrophobin, the distance of a protein-fused domain and the nanostructure are in the range of the FÖRSTER radius. It was successfully demonstrated that quantum dots and fluorescent proteins fulfill the spectroscopic requirements of such a donor/acceptor pair. The FRET performance of these excitable oil droplets was examined as a ‘proof of concept’. Due to its modular design, this signal amplification setup could be exploited in numerous fields of technical application ranging from quantification of micronutrient to photothermal cancer therapy.

During embryonic development, precursor germ cells contain aggregates of protein and RNA known as germ granules. These germ granules are important in the specifi- cation of a functioning germ line, i.e. functioning sex cells within mature organisms. In the single cell fertilized embryo of the nematode worm C.elegans, germ granules (referred to as P granules) localize to the posterior side of the cell. After cell division occurs, they are found only in the posterior daughter cell. The localization behav- ior of P granules has been a topic of much interest, and considered an important aspect of symmetry breaking during development. We learn the fundamental prop- erties of P granule localization, and determine possible parameters and features of this biological system by developing theory in close collaboration with experimental evidence. In this study, experimental evidence is presented which shows that P granules are liquid droplets, and that their localization occurs through preferential nucleation and growth behavior on one side of the cell and simultaneous preferential dissolution on the opposite side. It is also shown that this behavior is linked to the concentration gradient of the protein Mex-5 along the anterio-posterior axis of the cell, which is necessary to induce the preferential growth of P granules. From this experimental data, a theoretical model for the preferential growth of P granules is developed, where the localization of P granules occurs by phase separa- tion. That is, P granules separate from the bulk cytoplasm by a process described by a first order liquid-liquid phase transitition, where a liquid droplet granule phase nucleates and then grows out of the bulk liquid cytoplasmic phase. In this model, a spatial gradient is imposed on the saturation point, the boundary point between the single phase state consisting only of the cytoplasm, and a metastable state which includes both a P granule and cytoplasm phase. This gradient mimics the properties of the Mex-5 gradient and is sufficient in explaining P granule localization. Using numerical simulations, the theoretical model is studied. It is found suffi- cient to both successfully describe P granule localizaion, and to describe interesting behavior in a system with assymetric growth due to a spatial gradient. From a purely theoretical standpoint, we observe cyclical non-equilibrium steady states, where material is cycled back and forth along the gradient. From the biological side, experimental properties of the system, such as the diffusion coefficient of P granules and P granule growth rates are determined through both simulation and image analysis of data. In addition, the possiblility of different types of growth behavior at later cell stages, and a method of long range intracellular signalling are suggested from the theoretical model.

During vertebrate embryo development, the body axis is subdivided into repeated structures, called somites. Somites bud off from an un-segmented tissue called the pre-somitic mesoderm (PSM) in a sequential and periodic manner, tightly controlled by an in built molecular clock, called the "segmentation clock". According to current understanding, the clock is comprised of: (i) an autonomous cellular oscillator consisting of an intracellular negative feedback loop of Her genes within the PSM cells, (ii) Delta-ligand and Notch-receptor coupling that facilitates synchronization of oscillators among the PSM cells, (iii) Tissue level waves of gene expression that emerge in the posterior PSM and move coherently towards anterior, leading to global arrest of oscillations in the form of somites. However, the movement of cellular oscillators within the PSM before the formation of somitic furrows, a prominent feature of the tissue as observed through this work has not been experimentally considered as a constituent of the segmentation clock so far. Our work aims to incorporate movement of cellular oscillators in the framework of the segmentation clock. It is well known from theoretical studies that the characteristics of relative motion of oscillators affect their synchronization properties and the patterns of oscillations they form. Particularly, theoretical studies by Uriu et al., PNAS (2010) suggest that cell movements promotes synchronization of genetic oscillations. Here, we established experimental techniques and image analysis tools to attain quantitative insight on (i) diffusion co-efficient of cellular oscillators, (ii) dynamics of a population of oscillators, within the PSM aiming towards concomitant understanding of the relationship between movement and synchronization of cellular oscillators. In order to quantitatively relate cellular oscillators and their motion within the PSM, I established imaging techniques that enabled visualization of fluorescenctly labeled nuclei as readouts of cell positions in live embryo, and developed dedicated segmentation algorithm and implemented tracking protocol to obtain nuclei positions over time in 3D space. Furthermore, I provide benchmarking techniques in the form of artificial data that validate segmentation algorithm efficacy and, for the first time proposed the use of transgenic embryo chimeras to validate segmentation algorithm performance within the context of in vivo live imaging of embryonic tissues. Preliminary analysis of our data suggests that there is relatively high cell mixing in the posterior PSM, within the same spatial zone where synchronous oscillations emerge at maximum speed. Also, there are indications of gradient of cell mixing along the anterior-posterior axis of the embryo. By sampling single cell tracks with the help of nuclei markers, we have also been able to follow in vivo protein oscillations at single cell resolution that would allow quantitative characterization of coherence among a population of cellular oscillators over time. Our image analysis work flow allows testing of mutant embryos and perturbation of synchrony dynamics to understand the cause-effect relationship between movement and synchronization properties at cellular resolution. Essentially, through this work, we hope to bridge the time scales of events and cellular level dynamics that leads to highly coordinated tissue level patterns and thereby further our understanding of the segmentation clock mechanism.